Molecular phylogenetic study of several eelpout fishes (Perciformes,Zoarcoidei) from Far Eastern seas on the basis of the nucleotide sequences of the mitochondrial cytochrome oxidase 1 gene (<Emphasis Type="Italic">Co-1</Emphasis>)

A total of 95 nucleotide sequences of a Co-1 gene fragment of approximately 650 bp were analyzed for fishes of the orders Perciformes and Scorpaeniformes (outgroup). Gene trees based on four algorithms (BA, NJ, MP, and ML) were similar in topology of solved branches. An emphasis was placed on the species and generic levels, but a significant phylogenetic signal was obtained for higher taxonomic ranks as well. For instance, a monophyletic origin was confirmed for the family Zoarcidae and the subfamily Opisthocentrinae (Stichaeidae). The proportion of different nucleotides in the sequences compared (p-distances) significantly increased with increasing taxonomic rank. The p-distances were estimated for four hierarchic levels and were (1) 0.15 ± 0.06% for the within-species hierarchic level, (2) 6.33 ± 0.37% for the within-genus level, (3) 11.83 ± 0.06% for the within-family level, and (4) 15.22 ± 0.05% for the within-order level. The difference in the Co-1 gene fragments between levels (1) and (2) allows almost errorless species identification on the basis of this kind of a molecular bar code.     

Phylogenetic analysis of Altai osmans of the genus Oreoleuciscus (Pisces, Cyprinidae, Leuciscinae), based on the analysis of the cytochrome oxidase 1 gene (Co-1) sequence

Molecular genetic analysis of Altai osmans of the genus Oreoleuciscus from two different parts of the range was carried out. In this study, based on the mitochondrial Co-1 gene sequence, a total of 25 fish specimens belonging to four genera were examined: (1) O. humilis, 2 specimens; O. potanini, 13 specimens; (2) Pseudaspius leptocephalus, 1 specimen; (3) Tribolodon brandtii, T. hakonensis, and T. sachalinensis from the GenBank database, 8 speciens; and (4) Leuciscus waleckii, 1 specimen (used as an outgroup). The p-distances were very low both within and between the species: (1) 0.20 +/- 0.03%; (2) 0.40 +/- 0.12%; and (1-2) 0.80 +/- 0.04%. To visualize the relationships among all of the species examined, the neighbor joining (NJ), maximum parsimony (MP), Bayesian (BA), and maximum likelihood (ML) trees were constructed. The results obtained using these methods were very similar. It was demonstrated that species assignment of the individuals (barcoding) with the help Co-1 gene was effective, despite of very low divergence of the two osman taxa, which was comparable with typical intraspecific values in other animal groups. Taxonomic status of O. potanini and O. humilis requires further investigation with paying attention to low genetic distances between these species along with the lack of material from sympatric parts of the ranges.     

Phylogenetic and taxonomic analysis of flatfish species (Teleostei,Pleuronectiformes) inferred from the primary nucleotide sequence of cytochrome oxidase 1 gene (<Emphasis Type="Italic">Co-1</Emphasis>)

Seventeen nucleotide sequences of Co-1 gene from 13 Pleuronectiformes species and 2 Perciformes species served as the outgroup were examined. For divergence comparison, the initial stage involved calculation of pairwise p-distances for all investigated sequences. This allowed to evaluate the nucleotide diversity on four phylogenetically different levels: (1) intraspecific, (2) intrageneric, (3) intrafamilial, and (4) intraordinal. The values of p-distances for the Co-1 gene for the four mentioned categories were (1) 0.93 ± 0.73%, (2) 11.72 ± 1.86%, (3) 12.10 ± 1.10%, and (4) 20.20 ± 0.22%, respectively. An increase in the level of genetic divergence along with an enhancement in taxon rank was previously reported for different species, which might be explained by prevalence of geographic speciation model in nature. Phylogenetic trees were constructed using four approaches: maximum parsimony, Bayesian, maximum likelihood, and neighbor-joining. These trees demonstrated similar results confirming the monophyletic origin of the families studied. The examined representatives of the flatfish species and genera were shown to be sufficiently divergent genetically.     

Divergence at Cyt-b and Co-1 mtDNA genes on different taxonomic levels and genetics of speciation in animals

Genetic divergence estimates using p-distances and similar measures were generated for 20,731 vertebrate and invertebrate animal species. The results of this analysis demonstrate that the data series are realistic and interpretable when the p-distance and its various derivates are used. The focus is on vertebrates and fish species in particular and the newest data set. Distance data reveal increasing levels of genetic divergence of the sequences of the two genes, cytochrome b (Cyt-b) and cytochrome c oxidase subunit 1 (Co-1), in the five groups compared: populations within species; subspecies, semi-species, or/and sibling species; species within a genus; species from different genera within a family; and species from separate families within an order. Mean unweighted scores of p-distances (%) for these five groups are Cyt-b-1.38?±?0.30, 5.10?±?0.91, 10.31?±?0.93, 17.86?±?1.36, and 26.36?±?3.88, respectively; and Co-1-0.89?±?0.16, 3.78?±?1.18, 11.06?±?0.53, 16.60?±?0.69, and 20.57?±?0.40, respectively. The estimates show good correspondence with other analyses. These results testify to the applicability of p-distance for most intra-species and inter-species comparisons of genetic divergence up to the order level in animals for the two genes compared. Data reviewed provide empirical and theoretical background on the geographic speciation mode prevalence in species origin and give a framework why per-individual species identification (DNA barcoding) is usually successful.     

Sequence diversity at cytochrome oxidase 1 (Co-1) gene among sculpins (Scorpaeniformes, Cottidae) and some other scorpionfish of Russia Far East with phylogenetic and taxonomic insights

Mitochondrial DNA (mtDNA) atCo-1 gene region was sequenced for 7 scorpion fish species (in total, 16 sequences of at least 552 bp) from the Far East of Russia and compared with 15 other sequences of Scorpaeniformes comprising altogether 29 scorpion-like fish sequences and two outgroup sequences (Cypriniformes). The analysis of the protein-codingCo-1 gene revealed statistically substantiated bias in the (T+C) ∶ (A+G) content, proving basic findings. The average scores ofp-distances for different scales of the evolutionary history atCo-1 gene revealed a clear pattern of increased nucleotide diversity at four different levels: (1) intraspecies, (2) intragenus, (3) intrafamily, and (4) intraorder. The scores of averagep-distances for the compared fish groups were: (1) 1.00±0.20%, (2) 3.80±0.20%, (3) 12.40±1.20%, and (4) 18.00±0.38%, respectively (mean±SE). These data support the concept that speciation in the order Scorpaeniformes, in most cases, follows a geographic mode through accumulation of numerous small genetic changes over a long period of time. However, intraspecies diversity was surprisingly high among scorpionfish. Phylogenetic trees for 29 sequences of scorpionfish and 2 other fishes belonging to ray-finned fishes (Actinopterigii) were developed usingCo-1 gene and four different analytical approaches: Bayesian (BA), maximum likelihood (ML), neighbour-joining (NJ), and maximum parsimony (MP). The analysis revealed a monophyletic origin for the representatives of Cottidae, which is the principal scorpionfish family investigated (100, 96, 98% support level in our BA, MP, and NJ analyses). Similarly, the monophyletic origin of up to the three compared scorpion-like fish genera was supported by molecular phylogenetic data. Species identification on individual basis (barcoding tagging) was high. A few taxonomic complications arose during the analysis and they are discussed here in.     

Sequence divergence at mitochondrial genes in animals: applicability of DNA data in genetics of speciation and molecular phylogenetics

Nucleotide diversity estimates for the genes Cyt-b (cytochrome b) and Co-1 (cytochrome oxidase 1) are analyzed. Genetic divergence of populations (1) and taxa of different rank, such as subspecies, semispecies or/and sibling species (2), species within a genus (3), species from different genera within a family (4), and species from separate families within an order (5) have been compared using a database of p-distances and similar measures. Empirical data for 20,731 vertebrate and invertebrate animal species reveal various and increasing levels of genetic divergence of the sequences of the two genes, Cyt-b and Co-1, in the five groups compared. Mean unweighted scores of p-distances (%) for five groups are: Cyt-b (1) 1.38±0.30, (2) 5.10±0.91, (3) 10.31±0.93, (4) 17.86±1.36, (5) 26.36±3.88 and Co-1 (1) 0.89±0.16, (2) 3.78±1.18, (3) 11.06±0.53, (4) 16.60±0.69, (5) 20.57±0.40. These estimates testify to the applicability of p-distance for most intraspecies and interspecies comparisons of genetic divergence up to the order level for the two genes compared. The results of the analysis of the nucleotide divergence within species and higher taxa of animals suggest that a phyletic evolution in animals is likely to prevail at the molecular level, and speciation mainly corresponds to the geographic or divergence mode (type D1). The prevalence of the D1 speciation mode does not mean that other modes are absent. At least seven possible modes of speciation are considered. The approach suggested that allows recognize the speciation modes formally with the operational genetic criteria. Such approach may help to solve a key problem of the biological species concept, i.e. the lack of ability to monitor in most cases the reproductive isolation barriers between species.     

Complete mitochondrial genome of the bullhead torrent catfish, Liobagrus obesus (Siluriformes, Amblycipididae): Genome description and phylogenetic considerations inferred from the Cyt b and 16S rRNA genes

Mitochondrial DNA (mtDNA) from the bullhead torrent catfish, Liobagrus obesus, was isolated by long-polymerase chain reaction (Long-PCR) with universal primers and was fully sequenced by primer working using flanking sequences. The complete mtDNA from L. obesus was 16,531 bp in length and contained 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and a control region, demonstrating a structure very similar to that of other bony fish. An analysis of the protein-coding genes revealed a statistically substantiated bias in (T+C): (A+G) content, supporting earlier findings regarding this peculiarity. As indicated by a chi-square test, the observed scores for pyrimidine and purine content were different from those expected assuming a 50:50 ratio: chi(2)=41.63, d.f.=5, p<0.000001 for three categories, including the 1st, 2nd, and 3rd codon positions. Further, there was a difference in nucleotide content between ND6 and the other 12 protein-coding genes in L. obesus. The values of p-distances, as summarized for different scales of evolutionary history at the Cyt b gene, revealed a clear pattern of increased nucleotide diversity at four levels: (1) intraspecies, (2) intragenus, (3) intrafamily, and (4) intraorder. Scores of average p-distances of the four categories in catfish were (1) 1.59+/-0.54%, (2) 5.28+/-1.72% (3) 16.37+/-1.26%, and (4) 19.81+/-0.14%, respectively. These data support the hypothesis that speciation in the order Siluriformes, in most cases, follows a geographic mode through the accumulation of a numerous small genetic changes over a long time period. A phylogenetic tree for the bullhead torrent catfish and several other fish species belonging to the order Siluriformes was developed on the basis of respective Cyt b sequences (1138 bp); the analysis revealed a monophyletic origin for the five examined families. A species-specific clustering of sequences from single species was obtained, supporting additionally basic phylogenetic information for the catfish and the barcoding suitability of Cyt b sequence data. Lastly, one of the well-supported properties of our phylogenetic tree (99% repetition level in our analysis) was the monophyletic placement of all catfish (order Siluriformes) among other ray-finned fish of the class Actinopterigii. Also discussed herein are the aspects of phylogeny based on the 16S rRNA gene.     

Free access of published DNA sequences facilitates regular control of (meta-) data quality – an example from shorebird mitogenomes (Aves,Charadriiformes: Charadrius)

Online repositories of DNA sequences are a rich and indispensable source of comparative data for biodiversity research and taxonomic studies. Despite increasingly high data quality of published sequences and associated metadata, particular attention should be paid to taxonomic assignment of DNA sequences, in particular if voucher specimens are not available or cannot be examined. In this study, two nearly identical mitogenomes of two distinctive plover species (Charadrius alexandrinus and Charadrius placidus) were re-analysed and compared with a comprehensive dataset of DNA-barcode sequences (cytochrome-oxidase subunit 1, COI) for 55 shorebird species. Phylogenetic analysis separated the two plover species into two reciprocally monophyletic clades that differed by mean p-distances of 11.5–14.7%; however, the COI sequence from the C. placidus mitogenome was nested in the Kentish Plover clade (C. alexandrinus). A similar mismatch was found for another DNA-barcode sequence from a Charadrius mongolus mitogenome that clustered with one of two clades of Charadrius leschenaultii in the COI tree. These results strongly suggest that, to date, two of seven mitogenomes published for Charadriidae are not representative of the taxon names to which the respective GenBank entries were assigned. Only a few DNA-barcode sequences were associated with outdated taxonomy, while others were suspected to be chimeric sequences. Thus, free access to digital sequence information is a key factor for steady improvement of data quality in online repositories via swarm intelligence of the scientific community.     

Host specificity is linked to intraspecific variability in the genus Lamellodiscus (Monogenea)

SUMMARYWe investigated whether host specificity is linked to variability within species of Lamellodiscus monogeneans, which are gill ectoparasites of the Sparidae. We sampled fish parasites in the northeastern part of the Mediterranean Sea: 4 specialist species, using 1 single host species, and 3 generalist species, using 2 distinct host species. Intraspecific variability was assessed from 2 different datasets. Morphometric variability of the attachment organ, called the haptor, was estimated first from measurements of several sclerified haptoral parts on 102 individuals. Genetic variability was calculated based on comparisons of sequences derived from the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA of 62 individuals. Morphometric variances in the specialist versus generalist species were compared via principal component analysis and F-tests, and uncorrected genetic distances (p-distances) were estimated within each species. We showed that the inter-individual variance of morphometric characters, as well as p-distances, are clearly greater within generalist species than specialist ones. These findings suggest that a relative increase in morphological and molecular variability enhances the possibility to colonize new host species in monogeneans, and supports the hypothesis that intraspecific variability could be a potential determinant of host specificity.     

The first internal transcribed spacer (ITS-1) of Melipona species (Hymenoptera, Apidae, Meliponini): characterization and phylogenetic analysis

Summary. Internal transcribed spacer 1 (ITS-1) sequences of the nuclear rDNA of eight bee species of the genus Melipona were studied. Complete ITS-1 sequence and flanking regions from three Melipona species were PCR-amplified, cloned, sequenced, and their variability compared. These sequences show length variation (1391 to 1417 bp), several repeated elements of one, two, three, and four nucleotides, and a repeated tandem sequence of approximately 80 bp. The low variation level between M. quadrifasciata and M. mandacaia sequences supports the hypothesis that they diverged recently. PCR-amplification, cloning, and sequencing of a partial ITS-1 sequence (394 to 496 bp) of eight Melipona species and two outgroups were performed and the obtained sequences used for phylogenetic analysis. The single tree estimated from parsimony analysis recovered four well-defined clades and monophyly of the genus Melipona. The phylogenetic relationships derived from sequences of ITS-1 fragments corroborate the taxonomic classification of Melipona based on morphological characters.Received 17 July 2003; revised 10 May 2004; accepted 1 June 2004.     

Diversity and temporal variations of the Hemiptera Auchenorrhyncha fauna in the Ajaccio region (France,Corsica)

Summary

During a one-year survey of the Auchenorrhyncha fauna in a maquis habitat of the Ajaccio region (Corsica), 37 species were listed including three alien species recorded in Europe in the last decades. The standard 658 bp mitochondrial COI barcode was obtained for 32 species, including 14 species never previously barcoded. Neighbor-joining analyses confirmed the delineation for all the species. However, comparisons with available sequences from mainland Europe showed that four species presented a significant intraspecific divergence (>3%), whereas a reduced interspecific divergence was found for another set of species. Complementary studies may therefore be necessary to better assess their taxonomic levels. Three species – Euscelis lineolata Brullé, 1832 (Cicadellidae), Philaenus spumarius (Linnaeus, 1758) (Aphrophoridae) and Latilica maculipes (Melichar, 1906) (Issidae) – accounted for up to 74.3% of the total insects collected. Although taxonomic interpretations using restricted sampling and COI sequences alone are not robust, the proportion of new sequences highlight the global lack of knowledge about the Auchenorrhyncha as a whole and the potentially presence of cryptic taxa in different clades.     

Hiding deep in the trees: discovery of divergent mitochondrial lineages in Malagasy chameleons of the Calumma nasutum group

WE CONDUCTED A COMPREHENSIVE MOLECULAR PHYLOGENETIC STUDY FOR A GROUP OF CHAMELEONS FROM MADAGASCAR (CHAMAELEONIDAE: Calumma nasutum group, comprising seven nominal species) to examine the genetic and species diversity in this widespread genus. Based on DNA sequences of the mitochondrial gene (ND2) from 215 specimens, we reconstructed the phylogeny using a Bayesian approach. Our results show deep divergences among several unnamed mitochondrial lineages that are difficult to identify morphologically. We evaluated lineage diversification using a number of statistical phylogenetic methods (general mixed Yule-coalescent model; SpeciesIdentifier; net p-distances) to objectively delimit lineages that we here consider as operational taxonomic units (OTUs), and for which the taxonomic status remains largely unknown. In addition, we compared molecular and morphological differentiation in detail for one particularly diverse clade (the C. boettgeri complex) from northern Madagascar. To assess the species boundaries within this group we used an integrative taxonomic approach, combining evidence from two independent molecular markers (ND2 and CMOS), together with genital and other external morphological characters, and conclude that some of the newly discovered OTUs are separate species (confirmed candidate species, CCS), while others should best be considered as deep conspecific lineages (DCLs). Our analysis supports a total of 33 OTUs, of which seven correspond to described species, suggesting that the taxonomy of the C. nasutum group is in need of revision.     

Genealogical exclusivity in geographically proximate populations of Hypochilus thorelli Marx (Araneae,Hypochilidae) on the Cumberland Plateau of North America

The issue of sampling sufficiency is too infrequently explored in phylogeographical analysis, despite both theoretical work and analytical methods that stress the importance of sampling effort. Regarding the evolutionary pattern of reciprocal monophyly, both the probability of recovering this pattern and the possible inferences derived from this pattern, are highly contingent upon the density and geographical scale of sampling. Here, we present an empirical example that relates directly to this issue. We analyse genetic structure in the southern Appalachian spider Hypochilus thorelli, using an average sample of 5 mitochondrial DNA (mtDNA) sequences per location for 19 locations. All sampled sites are reciprocally monophyletic for mtDNA variation, even when separated by geographical distances as small as 5 km. For populations separated by greater geographical distances of 20-50 km, mtDNA sequences are not only exclusive, but are also highly divergent (uncorrected p-distances exceeding 5%). Although these extreme genealogical patterns are most seemingly consistent with a complete isolation model, both a coalescent method and nested cladistic analysis suggest that other restricted, but nonzero, gene flow models may also apply. Hypochilus thorelli appears to have maintained morphological cohesion despite this limited female-based gene flow, suggesting a pattern of stasis similar to that observed at higher taxonomic levels in Hypochilus.     

Species delimitation through DNA barcoding of freshwater leeches of the Glossiphonia genus (Hirudinea: Glossiphoniidae) from Eastern Siberia,Russia

The study of biodiversity is a priority task of biological science. The structural unit of biodiversity is a species that has a clear identification in a taxonomic system. Morphological features are traditionally the main criteria for species discrimination in zoological studies. However, the presence of inter- and intraspecific polymorphism and phenotypic plasticity makes it difficult to identify species in many groups of invertebrates. To solve this problem, in this research, we analyzed morphological and genetic data in combination to delimit species among the Eastern Siberia Glossiphonia leeches using different approaches. Morphology analysis revealed phenetically distinct groups, suggesting the existence of at least two species in the region, G. verrucata, a rare Palaearctic species, and a potentially new species Glossiphonia sp. Moreover, sequence-based species delimitation methods congruently supported eight distinct species groups (including two Siberian species) within the available molecular dataset of the Glossiphonia world fauna, using phylogenetic (ML and BI), coalescent (ABGD and GMYC) methods, and pairwise analysis of sequences. The detected p-distances (modal value of 0.11) between these 8 groups and the level of genetic polymorphism (max. 0.0041) within groups indicate that the groups are 8 independent species according to the DNA barcoding. Our results once again proved the usefulness of molecular systematics. At the same time, we detected several inaccuracies in the leech species identification, as well as many ambiguous sites in sequences uploaded on GenBank, which affects the analysis and impedes progress of DNA barcoding technology.     

中国蛛缘蝽科物种DNA条形码研究(英文)

【目的】本研究旨在探讨DNA条形码对中国蛛缘蝽科(半翅目：缘蝽总科)物种界定的适用性。【方法】对中国蛛缘蝽科13属23种207个样本的线粒体COI基因DNA条形码序列进行扩增,并扩增稻缘蝽属Leptocorisa 3个物种的31条内转录间隔区1(ITS-1)序列作为辅助标记。使用MEGA 11软件计算种间和种内遗传距离(Kimura 2-parameter, K2P);采用邻接法(neighbor-joining, NJ)进行物种聚类分析;利用中介邻接网络算法构建单倍型网络图。【结果】基于线粒体COI DNA条形码序列得出测试的中国蛛缘蝽科所有23个种的种内平均K2P距离在2%以下,种间K2P距离在0.98%~23.98%之间(平均17.50%)。多数物种彼此能够被较好地分开,且支持率较高。其中,中稻缘蝽Leptocorisa chinensis和大稻缘蝽L. oratoria共享部分COI单倍型,造成COI条形码无法区分二者,可通过ITS-1序列在单倍型网络分析中将二者区分。【结论】本研究得出的中国蛛缘蝽科中绝大部分物种的DNA条形码数据分析结果与基于形态特征的分类单元一致。然而,对于其中亲缘关系极近的物种,单靠线粒体数据尤其是COI条形码序列无法进行准确界定,需引入其他DNA序列或其他类型数据进行区分。     

Numerical taxonomic studies on Solarium L. section Solarium (Maurella)

Population differentiation was studied in 32 accessions belonging to Solarium L. section Solarium (Maurella); these accessions represented 20 species of four differing ploidy levels. A study of discordance demonstrated that the species exhibit considerable inherent discordant variation; it also provided an estimate of the relative variabilities of characters within these populations, and the consistency with which they differentiate populations.
Hierarchic clusters, obtained using the single-link cluster method, confirmed the relative importance of the different classes of characters used for differentiation within this section. Similarly, the hierarchic clusters generally supported earlier taxonomic decisions.
The use of a non-hierarchic cluster method provided the most interesting data on differentiation within the section Solarium, particularly for genome relationships of the polyploid taxa. This clustering pattern fitted well with known genome relationships, while also suggesting putative ancestors for other allopolyploids; suggestions deriving retrospective support from the hierarchic dendrograms. The progenitors suggested by tliese two clustering methods supported data derived from orthodox taxonomy, comparative morphology, hybridisation studies, the artificial synthesis of polyploids and seed protein band patterns.     

Phylogenetic relationships of the marine Haplosclerida (Phylum Porifera) employing ribosomal (28S rRNA) and mitochondrial (cox1, nad1) gene sequence data

The systematics of the poriferan Order Haplosclerida (Class Demospongiae) has been under scrutiny for a number of years without resolution. Molecular data suggests that the order needs revision at all taxonomic levels. Here, we provide a comprehensive view of the phylogenetic relationships of the marine Haplosclerida using many species from across the order, and three gene regions. Gene trees generated using 28S rRNA, nad1 and cox1 gene data, under maximum likelihood and Bayesian approaches, are highly congruent and suggest the presence of four clades. Clade A is comprised primarily of species of Haliclona and Callyspongia, and clade B is comprised of H. simulans and H. vansoesti (Family Chalinidae), Amphimedon queenslandica (Family Niphatidae) and Tabulocalyx (Family Phloeodictyidae), Clade C is comprised primarily of members of the Families Petrosiidae and Niphatidae, while Clade D is comprised of Aka species. The polyphletic nature of the suborders, families and genera described in other studies is also found here.